Processes for the purification of proteins are generally known and include such techniques as ion exchange chromatography, adsorption chromatography, gel electrophoresis, ammonium sulfate precipitation, and gel filtration.
Although each of these techniques is known, it is impossible to predict the extent to which any of the above-listed techniques is applicable to the purification of a given protein. Various factors including the extent of purification desired, the extent of acceptable loss of biological activity of the protein, and degree of homogeneity of the protein desired, require extensive experimentation to optimize the purification of the products.
Human TNF has been purified as a native protein from culture supernatants of induced HL-60 cells by a combination of anion exchange chromatography and reverse phase high pressure liquid chromatography (HPLC), with elution in a linear gradient of acetonitrile (Wang, A. M., et al., Science (1985) 228:149-154). Similar procedures had been previously employed (Matthews, N., Br. J. Cancer (1981) 44:418) without resulting in a homogeneous preparation. However, this technique is not optimally efficient even for the native TNF secreted from, for example, HL-60 or other TNF secreting cell lines, and is inappropriate for recombinantly produced TNF, due to substantial inactivation of TNF biological activity at low pH.
Copending U.S. application Ser. No. 792,815 filed Oct. 20, 1985 now U.S. Pat. No. 4,677,197 and assigned to the same assignee of the present application improves upon the process of Wang et al. supra. Whereas Wang et al. obtain a product that is not homogeneous by the steps of anion exchange chromatography, followed by HPLC and elution in a linear acetonitrile gradient, U.S. application Ser. No. 792,815 achieves an active homogeneous recombinantly produced TNF product. In the improved process according to U.S. application Ser. No. 792,815 a hydrophobic support is substituted for the reverse phase HPLC of Wang, M. et al., supra and Matthews, N. et al., supra.
European Patent Publication No. 168,214 published Jan. 15, 1986 discloses a process for purifying TNF by the steps of obtaining a TNF solution from cell culture supernatants or lysates, removing solids, adsorbing TNF from the remaining supernatant onto a silicate support, eluting TNF from the silicate support, chromatographing TNF on a tertiary amino anion exchange resin, and chromatographing TNF on an anion exchange resin containing quaternary ammonium substituents. Optional purification steps including chromatofocusing to concentrate and purify the product or passage through a sieving gel such as Sephadex G-25 are disclosed. As a hydrophobic support, EP Publication 168,214 discloses the use of silicate, polyolefin and alkyl Sepharose. The TNF is eluted from the silicate using a polyol, preferably ethylene glycol in a 10-30% range, with a 20% (v/v) concentration preferred. Further purification, according to the process, requires adsorption onto a tertiary or quaternary amino anion exchange resin such as DEAE cellulose, QAE Sephadex or the product sold under the tradename Mono Q. Purification to homogeneity, according to the process, is accomplished only upon further separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or C4 reverse phase high performance liquid chromatography (HPLC). These latter steps are accompanied, however, by substantial loss of biological activity.
The present invention provides a method for purifying TNF that produces a substantially homogeneous TNF without recourse to reverse phase HPLC or SDS-PAGE electrophoresis. The method disclosed is applicable to large scale purification of TNF. When the host cell is a Gram-negative microorganism such as E. coli and the TNF is thus recombinantly produced, a number of host cell proteins and other substances are produced with the TNF. Such co-produced materials include endotoxins and pyrogenic materials that must be selectively removed from the TNF. The use of a filtration step through a hydrophobic porous matrix, offers substantial recovery of active TNF proteins and substantially complete removal of host cell proteins, endotoxins and pyrogens when the host cell is a Gram-negative microorganism, before the use of any chroaatographic techniques in purifying the TNF. As a result, large scale recovery of the material is possible.